Compositions, methods and therapies for administering antigen peptide

ABSTRACT

The invention relates to compositions, methods and therapies for the treatment of inflammation caused by infection with  Propionibacterium acnes . The compositions include a combination of peptide and anti-TNF. The peptide consists of a specific amino acid sequence or a peptide consisting of an amino acid sequence derived the specific amino acid sequence by deletion, substitution, insertion or addition of one or more amino acids. The administration of the peptide and anti-TNF in therapeutically effective amounts to a patient is effective to suppress, by immune response, inflammation caused by infection with  Propionibacterium acnes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application and claims priorityfrom continuation-in-part U.S. patent application Ser. No. 14/312,162,filed Jun. 23, 2014, entitled “Compositions, Methods and Therapies forAdministering Antigen Peptide”, and from U.S. patent application Ser.No. 13/380,425 filed Dec. 22, 2011, entitled “Antigen Peptide and UseThereof”, from PCT International Application PCT/JP2011/062305 entitled“Antigen Peptide and Use Thereof” filed May 23, 2011, and fromProvisional Application 61/396,574, entitled “Antigen Peptide and UseThereof” filed on May 27, 2010. These references are hereby incorporatedin their entirety by reference.

FIELD OF THE INVENTION

The invention relates generally to antigen peptides and their useagainst Propionibacterium acne. The invention further includes thecombination of antigen peptides and anti-tumor necrosis factor(anti-TNF), and the use thereof against Propionibacterium acne. Theinvention also relates to methods and therapies for treating a patienthaving Propionibacterium acne. More particularly, the invention includesadministering antigen peptide and anti-TNF to a patient intherapeutically effective amounts.

BACKGROUND OF THE INVENTION

Acne is a known inflammatory disease that often appears on portions of apatient extending from the neck to the face of the human body.Typically, acne is most prevalent during puberty, however, it is knownthat the presence of acne can be found during adulthood as well. Due tothe inflammation occurring on portions of the human body which areexposed and visible, acne may have a significant psychological influenceon the person inflicted with this disease.

Various therapeutic agents for acne have been developed. In particular,there have been drugs developed which directly act on Propionibacteriumacnes that is a pathogen of acne.

In the art, the complete genome sequence of Propionibacterium acnes hasbeen determined and as a result, it is known that many genes ofPropionibacterium acnes encode a virulence factor. Based on thisinformation, research on sialidase of Propionibacterium acnes has beencarried out in developing a vaccine.

Tumor necrosis factor (TNF) is a cytokine which circulates throughoutthe body. TNF is critical for effective immune surveillance and isrequired for proper proliferation and function of natural killer cells,T cells, B cells, macrophages, and dendritic cells. The primary role ofTNF is in the regulation of immune cells. It is known that TNF can causesystemic inflammation which can result in various chronic conditions.Anti-TNF, also known as TNF blockers or inhibitors, interferes with thebody's production of TNF and as a result, is effective to reduceinflammation.

Most drugs that have been developed to treat Propionibacterium acnes acton bacterial cells of Propionibacterium acnes that are a cause of acneand exert an anti-inflammatory effect. Accordingly, in a situation wherethere is reinfection with Propionibacterium acnes, inflammation occursagain. Thus, the use of such drugs may be effective temporarily, buttheir use induces the emergence of a drug-resistant bacterium whichmakes treatment more difficult.

There is a need in the art to develop antigen peptides againstPropionibacterium acnes, as well as therapies and methods ofadministering the antigen peptides to regress, reduce or eliminatePropionibacterium acnes in a patient, and more particularly, therapiesand methods which enable the continuous treatment of acne. It isdesirable for the therapies and methods of administration to beeffective in a reasonable period of time. A continuous treatment of acneincludes immunotherapy, that is, a vaccine. Without intending to bebound by any particular theory, it is believed that establishingeffective anti-acne immunity (recognition and immunologic memory ofspecific Propionibacterium acnes), can result in the immediateelimination of Propionibacterium acnes at least on reinfection withstrains of the same Propionibacterium acnes. Accordingly, it isreasonably expected that immunotherapy can exert a continuousanti-inflammatory effect.

In accordance with the invention, it has been found that a peptidesconsisting of an amino acid sequence and anti-TNF can efficientlysuppress inflammation induced by Propionibacterium acnes in patients.

SUMMARY OF THE INVENTION

In one aspect, the invention solves the above needs by providing acomposition which includes peptide and anti-TNF. The peptide includesone or more of: (a) a peptide consisting of an amino acid sequenceindicated by SEQ NO. 1, (b) a peptide consisting of an amino acidsequence derived from the amino acid sequence indicated by SEQ NO. 1 bydeletion, substitution, insertion, or addition of one or several aminoacids, (c) a peptide consisting of an amino acid sequence indicated bySEQ NO. 3, and (d) a peptide consisting of an amino acid sequencederived from the amino acid sequence indicated by SEQ NO. 3 by deletion,substitution, insertion, or addition of one or several amino acids.

The peptide present in the composition can include: (e) a peptide whichis in a form of a multivalent antigen peptide obtained by binding aplurality of peptides described in the above (a) or (b) by a linker, and(0 a peptide which is in a form of a multivalent antigen peptideobtained by binding a plurality of peptides described in the above (c)or (d) by a linker.

In another aspect, the invention provides a method of suppressinginflammation caused by infection with Propionibacterium acnes in apatient. The method includes introducing into the patient peptide andanti-TNF. The peptide includes one or more of: (a) a peptide consistingof an amino acid sequence indicated by SEQ NO. 1, (b) a peptideconsisting of an amino acid sequence derived from the amino acidsequence indicated by SEQ NO. 1 by deletion, substitution, insertion, oraddition of one or several amino acids, (c) a peptide consisting of anamino acid sequence indicated by SEQ NO. 3, and (d) a peptide consistingof an amino acid sequence derived from the amino acid sequence indicatedby SEQ NO. 3 by deletion, substitution, insertion, or addition of one orseveral amino acids.

In certain embodiments, the peptide and anti-TNF are introducedsimultaneously into the patient. The peptide and the anti-TNF can becombined to form a composition and the composition can be introducedinto the patient. In other embodiments, the peptide and the anti-TNF canbe introduced separately into the patient. The peptide and the anti-TNFcan be introduced sequentially into the patient. Either one of thepeptide or the anti-TNF can be introduced into the patient, followed bythe introduction of the other one.

In another aspect, the invention provides a vaccine againstPropionibacterium acnes, including the above peptide and anti-TNF.

In another aspect, the invention provides a polynucleotide encoding theabove peptide.

In another aspect, the invention provides an expression vector to whichthe above polynucleotide is operably linked. Use of the expressionvector of the invention enables providing a peptide which can suppressinflammation caused by infection with Propionibacterium acnes.

In another aspect, the invention provides an antibody which specificallyrecognizes the above peptide.

In another aspect, the invention provides a method for determiningeffectiveness of a vaccine against Propionibacterium acnes. The methodincludes the step of detecting whether a polynucleotide encoding theabove peptide exists in a sample gathered from a living body. Use of themethod of the invention enables ease of determining whether the vaccineagainst Propionibacterium acnes is effective in a living body or not,before administering the vaccine to the living body.

In another aspect, the invention provides a method for treating orpreventing inflammation caused by Propionibacterium acnes includes thestep of administering to an individual the above vaccine, the vaccinecontaining a therapeutically effective amount of the peptide andanti-TNF.

In another aspect, the invention provides a method for treating orpreventing inflammation caused by Propionibacterium acnes including thestep of administering to an individual the above vaccine, the vaccinecontaining a therapeutically effective amount of the polynucleotide andanti-TNF.

BRIEF DESCRIPTION OF DRAWINGS

A further understanding of the invention can be gained from thefollowing description of the preferred embodiments when read inconjunction with the accompanying drawings in which:

FIG. 1 is a diagram showing a result of measuring inflammatory areascaused by injection of Propionibacterium acnes into individual portions;

FIG. 2 is a diagram showing a relation between an inflammatory areacaused by injection of Propionibacterium acnes and elapsed time afterthe injection;

FIG. 3 is a diagram illustrating steps of examining anti-inflammatoryeffects of peptides;

FIG. 4 is a diagram showing a result of examining anti-inflammatoryeffects of peptides;

FIG. 5 is a diagram showing another result of examininganti-inflammatory effects of peptides;

FIG. 6 is a diagram comparing a 16S rDNA base sequence of a strainisolated from a patient and a 16S rDNA base sequence of a standardstrain;

FIG. 7 is a diagram showing a result of examining anti-inflammatoryeffects of a peptide against injection of Propionibacterium acnesisolated from a patient;

FIG. 8 is a diagram showing photos of follow-ups of an acne patientvaccinated with a vaccine in accordance with certain embodiments of theinvention;

FIG. 9 is a diagram showing photos of follow-ups of another acne patientvaccinated with a vaccine in accordance with certain embodiments of theinvention;

FIG. 10 is a diagram showing photos of follow-ups of still another acnepatient vaccinated with a vaccine in accordance with certain embodimentsof the invention; and

FIG. 11 is a diagram showing photos of follow-ups of yet another acnepatient vaccinated with a vaccine in accordance with certain embodimentsof the invention.

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard letter abbreviations and the sequence listing isincorporated by reference herein.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions, methods and therapies to addressPropionibacterium acnes in a patient and the inflammation resultingtherefrom. The compositions, methods and therapies include the use ofantigen peptide. In certain embodiments, the antigen peptide isadministered in combination with anti-TNF. The antigen peptide with orwithout the presence of anti-TNF, is capable of inducing an immuneresponse against Propionibacterium acnes infection and reducing orpreventing inflammation caused by Propionibacterium acnes. The antigenpeptide of the invention has a very high antigenicity. Therefore, use ofthe peptide with or without anti-TNF can provide a vaccine againstPropionibacterium acnes.

In certain embodiments, use of peptide in combination with anti-TNF canproduce enhanced or improved results relative to reducing or eliminatingthe inflammation resulting from Propionibacterium acnes. The anti-TNFfor use in accordance with the invention can be in various forms. Forexample, the anti-TNF can be incorporated into a delivery mechanism,such as a liquid carrier or medium, to facilitate introduction such as,by injection.

The anti-TNF for use in accordance with the invention may include a widevariety of suitable materials. Non-limiting examples include anymaterial which demonstrates an anti-TNF effect or result, e.g., reducesinflammation generated by TNF. Further, suitable anti-TNF materials foruse in accordance with the invention can be selected from a wide varietyof anti-TNF materials known in the art. In certain embodiments, a TNFinhibitor including a fusion protein, such as but not limited to,Entanercept may be used. Entanercept is commercially available under thetrade name Enbrel.

The peptide for use in accordance with the invention may be one or moreof peptides (a) to (d) as follows:

(a) a peptide consisting of an amino acid sequence indicated by SEQ NO.1;

(b) a peptide consisting of an amino acid sequence derived from theamino acid sequence indicated by SEQ NO. 1 by deletion, substitution,insertion, or addition of one or several amino acids, the peptidegenerating, by immune response, an antibody suppressing an increase ofPropionibacterium acnes;

(c) a peptide consisting of an amino acid sequence indicated by SEQ NO.3; and

(d) a peptide consisting of an amino acid sequence derived from theamino acid sequence indicated by SEQ NO. 3 by deletion, substitution,insertion, or addition of one or several amino acids, the peptidegenerating, by immune response, an antibody suppressing an increase ofPropionibacterium acnes.

Here, the “several amino acids” can be, for example, two or three aminoacids, and more preferably, two amino acids.

In an embodiment wherein an amino acid is substituted, preferablesubstitution is substitution of one amino acid for another amino acidthat has the same properties as the one amino acid, such as substitutionof one amino acid for another amino acid that has the same side-chainfunctional group as the one amino acid (e.g., substitution of Arg forLys and substitution of Asp for Glu); and substitution of onehydrophobic amino acid for another hydrophobic amino acid (e.g.,substitution of Val for Ile).

As used herein and the claims, the term “peptide” is used in a mannerexchangeable with “polypeptide” or “protein”.

The peptide consisting of the amino acid sequence indicated by SEQ NO. 1corresponds to amino acid residues from 90th amino acid residue to 103rdamino acid residue in a membrane protein of Propionibacterium acneswhich consists of an amino acid sequence registered in Genbank/EMBL/DDBJwith the Accession No. AAT84059. The peptide consisting of the aminoacid sequence indicated by SEQ NO. 3 corresponds to amino acid residuesfrom 260th amino acid residue to 272nd amino acid residue in a membraneprotein of Propionibacterium acnes which consists of an amino acidsequence registered in Genbank/EMBL/DDBJ with the Accession No.YP_056445.

In general, in the production of antibodies, an amino acid sequencewhich is excellent as an antigen is a hydrophilic amino acid sequenceand an amino acid sequence having abundant charges, and also an aminoacid sequence that is predicted to have a turn structure. The chargesmay be either positive or negative and it is desirable to include atleast five charged amino acids. Further, the length of a selected aminoacid preferably corresponds to 10 to 15 residues. The amino acidsequence indicated by SEQ NO. 1 or 3 is selected accordingly.

These peptides induce immunity to Propionibacterium acnes that isxenobiotic. It is inferred that at the induction of immunity, productionof specific antibody is induced as a result of induction of Th2dominated immunity.

Methods of producing peptides in accordance with the invention canemploy chemical synthesis or an expression vector. In the embodimentswhich employ chemical synthesis, the peptide of the invention can beproduced by well-known methods of synthesis. Non-limiting examplesinclude but are not limited to liquid peptide synthesis and solid phasepeptide synthesis. In the embodiments which employ an expression vector,peptide may be produced from a transformant to which an expressionvector has been introduced or may be produced using an in vitrotranslation system. For example, a polynucleotide encoding the peptide(e.g., polynucleotide including a base sequence indicated by SEQ NO. 2or 4) is inserted into an expression vector and the expression vector isintroduced into a host cell. A target peptide can be produced in thehost cell. The term “transformant” used herein and the claims indicatesnot only a cell, a tissue, and an organ, but also a living organismitself. Methods for preparing a transformant include procedures that arewell known in this field, such as transformation by introducing arecombinant vector into a host. Non-limiting examples of livingorganisms to be transformed include, but are not limited to,microorganisms, plants, and animals. Introduction of a gene into a hostcell may be confirmed by a variety of procedures well known in thisfield, such as but not limited to PCR, Southern hybridization andNorthern hybridization.

In the embodiments wherein the peptide of the invention is produced bypreparing a transformant, it is preferable that the peptide is expressedstably in a host cell. However, the peptide may be expressed transientlyin a host cell. The peptide thus produced may be purified by a knownmethod. Non-limiting examples of suitable methods for purifying thepeptide include, but are not limited to, gel filtration chromatography,ion exchange chromatography, and affinity chromatography.

In another aspect, the peptide of the invention may be in the form of amultivalent antigen peptide (which may be also referred to as MAP:Multiple Antigen Peptide) obtained by binding a plurality of peptides(a) or (b) or by binding a plurality of peptides (c) or (d) below via alinker, as follows:

(a) a peptide consisting of an amino acid sequence indicated by SEQ NO.1;

(b) a peptide consisting of an amino acid sequence derived from theamino acid sequence indicated by SEQ NO. 1 by deletion, substitution,insertion, or addition of one or several amino acids, the peptidegenerating, by immune response, an antibody suppressing an increase ofPropionibacterium acnes;

(c) a peptide consisting of an amino acid sequence indicated by SEQ NO.3; and

(d) a peptide consisting of an amino acid sequence derived from theamino acid sequence indicated by SEQ NO. 3 by deletion, substitution,insertion, or addition of one or several amino acids, the peptidegenerating, by immune response, an antibody suppressing an increase ofPropionibacterium acnes.

MAP is a peptide capable of showing a higher antigenicity and is amultivalent peptide obtained by adding a linker to a C-terminus of aspecific peptide, and binding via the linker the specific peptide with aMAP structure (matrix) constructed based on Lys. MAP may be synthesizedby conventional and known methods. The number of specific peptides to beincluded in one MAP molecule is not particularly limited, but ispreferably five or more. The upper limit of the number of specificpeptides to be included in one MAP molecule is not limited since thenumber is preferably large in terms of antigenicity. In certainembodiments, restriction may be due to synthesis technique andtherefore, MAP may be produced with the number of specific peptidesbeing fifteen molecules or less.

In general, a Cys residue is used as an amino acid constituting a linkerto be added to a specific peptide. However, in the peptide in the formof MAP in accordance with certain embodiments of the invention, there isused a linker including a Gly residue between a specific peptide and aCys residue. Specifically, -GlyCys or -GlyGlyCys is added as a linker toa C-terminus of the specific peptide. For example, -GlyCys is added to aC-terminus of a peptide consisting of the amino acid sequence indicatedby SEQ NO. 1, and -GlyGlyCys is added to a C-terminus of a peptideconsisting of the amino acid sequence indicated by SEQ NO. 3. Since theGly residue is added between the Cys residue and the C-terminus of thespecific peptide, the Cys residue is given flexibility in rotation sothat when binding to the MAP structure, the specific peptide does notsuffer steric hindrance and the Cys residue at the linker can form anangle which enables the Cys residue to be close to a MAP reactive group.The linker for the peptide of the invention in the form of MAP is notlimited by this linker, and may be a general linker constituted by theCys residue.

In another aspect, the peptide of the invention is provided in the formof a peptide composition containing peptide (e) or (f), as follows:

(e) a peptide in the form of a multivalent antigen peptide obtained bybinding a plurality of the peptides (a) or (b) via a linker; and

(f) a peptide in the form of a multivalent antigen peptide obtained bybinding a plurality of the peptides (c) or (d) via a linker.

The term “peptide composition” used herein and the claims indicates asubstance containing a plurality of peptide components.

Individual peptide components can be combined in various ratios to formthe peptide composition. The ratios of individual components is notparticularly limiting and may be 1:1 (weight ratio), for example.Further, to ensure higher immunogenicity, the peptide compositionpreferably includes a peptide in the form of a multivalent antigenpeptide obtained by binding via a linker a plurality of peptidesconsisting of the amino acid sequence indicated by SEQ NO. 1, and apeptide in the form of a multivalent antigen peptide obtained by bindingvia a linker a plurality of peptides consisting of the amino acidsequence indicated by SEQ NO. 3.

Further, the invention provides a polynucleotide encoding the peptides(a)-(d). Non-limiting examples of the polynucleotide of the inventioninclude, but not limited to, a polynucleotide consisting of a basesequence indicated by SEQ NO. 2 or 4. The term “base sequence” as usedherein is exchangeable with “nucleic acid sequence” or “nucleotidesequence”, and is indicated as a sequence of deoxyribonucleotides(abbreviated as A, G, C, and T). The base sequence indicated by SEQ NO.2 encodes the peptide consisting of the amino acid sequence indicated bySEQ NO. 1, and the base sequence indicated by SEQ NO. 4 encodes thepeptide consisting of the amino acid sequence indicated by SEQ NO. 3.

The polynucleotide of the invention may be in the form of DNA or RNA. Aperson skilled in the art can easily produce the polynucleotide of theinvention based on amino acid sequence information of the peptide of theinvention and base sequence information encoding the peptide.Specifically, the polynucleotide of the invention can be produced bygeneral DNA synthesis or PCR.

Further, the invention provides a vector containing the polynucleotideencoding the peptide. When the vector is used in production of thepeptide, it is preferable that the vector is an expression vector towhich the polynucleotide is operably linked. The phrase “operably linkedto” as used herein and the claims indicates that a polynucleotide forencoding a target peptide is under the control of a control region suchas a promoter and is capable of expressing the peptide in a host cell. Aprocedure for causing a polynucleotide encoding a target peptide to be“operably linked to” an expression vector so as to construct a desiredvector is well known in the art. Further, the technique of introducingan expression vector into a host cell is also well known in the art.Accordingly, a person skilled in the art can easily produce a desiredpeptide in a host cell.

Use of the peptide of the invention and/or the polynucleotide of theinvention enables inducing immunity to Propionibacterium acnes. Further,the peptide and/or polynucleotide of the invention in combination withanti-TNF enables inducing immunity to Propionibacterium acnes. That is,the peptide and/or peptide/anti-TNF combination of the invention and/or,the polynucleotide and/or polynucleotide/anti-TNF combination of theinvention can serve as vaccines against Propionibacterium acnes.Accordingly, use of the peptide and/or peptide/anti-TNF combination ofthe invention and/or the polynucleotide and/or polynucleotide/anti-TNFcombination of the invention enables subduing intradermic inflammation(e.g., acne and pimples) due to Propionibacterium acnes.

The invention also includes a vaccine against Propionibacterium acnesand contains the aforementioned peptide and/or peptide/anti-TNFcombination, the aforementioned peptide composition and/or peptidecomposition/anti-TNF combination, or the aforementioned polynucleotideand/or polynucleotide/anti-TNF combination as an effective component.The term “vaccine” used herein indicates a vaccine which is used forimmunotherapy (vaccine therapy) and which induces an immune responsespecific to the peptide of the invention.

The vaccine of the invention may be provided in the form of a vaccinecomposition or in the form of a vaccine kit. Herein, “a vaccinecomposition containing an effective component” and “a vaccine kitcontaining an effective component” are generically referred to as “avaccine containing an effective component”. “Kit” indicates a form inwhich individual components are contained in different substances suchthat at least one component is contained in one substance and the restis contained in another substance. The vaccine composition and thevaccine kit will be detailed later herein.

In general, the term “vaccine composition” indicates “a vaccinesubstance in which two or more components exist as a whole so that thecomponents are considered as one substance”. Administration of thevaccine of the invention may be, for example, single administration ofpeptide or peptide and anti-TNF wherein the peptide consists of theamino acid sequence indicated by SEQ NO. 1 or the amino acid sequenceindicated by SEQ NO. 3, or a mixture of these two peptides. In theembodiments wherein a mixture of two peptides is employed, even if anamino acid sequence of an antigen protein of Propionibacterium acneswhich antigen protein corresponds to one of the two peptides is mutatedand the anti-inflammatory effect of the one peptide is lost, the otherpeptide can exert the anti-inflammatory effect. Such a composition maycontain other components (e.g., support which is pharmaceuticallyacceptable) in addition to the peptides and anti-TNF.

In general, when the peptide, anti-TNF and any other components are notcombined into the vaccine composition, a combination of individualcomponents cannot be recognized as one composition, but can beencompassed by the term “kit” and can be provided as a kit. This isreadily understood by a person having ordinary skill in the art.

In certain embodiments, the invention provides a vaccine compositionincluding the peptide of the invention and/or peptide and anti-TNF. Inother embodiments, the vaccine composition contains the polynucleotideof the invention and/or polynucleotide and anti-TNF. The polynucleotideis preferably in a form capable of expressing the peptide encoded by thepolynucleotide of the invention, and is more preferably provided in aform of an expression vector to which the polynucleotide of theinvention is operably linked. As described above, the peptide of theinvention or the polynucleotide of the invention is used as an effectivevaccine component in preparing a vaccine against Propionibacteriumacnes.

The vaccine of the invention can be used as a prophylactic vaccine or atherapeutic vaccine against Propionibacterium acnes. The term“prophylactic vaccine” as used herein and the claims indicates a vaccineadministered to an un-infected and untreated individual to prevent theindividual from being infected with a disease. The term “therapeuticvaccine” as used herein and the claims indicates a vaccine administeredto an individual already infected with a disease to improve theinfection or make the infection as small as possible or to eliminate theimmunopathological effect of the disease.

Various conventional methods for preparing vaccines containing aneffective component is well known by a person skilled in the art and maybe employed to prepare the vaccines in accordance with the invention.The vaccines may be prepared in the form of a solution or a suspensionsuch that they are injectable into the patient. Alternatively, thevaccine of the invention may be prepared in an appropriate solid form tobe solved or suspended in a solution prior to injection into thepatient. Such preparation may be emulsified or may be protein emulsifiedin a ribosome.

The effective component(s) of the vaccine composition of the inventionmay be mixed with a diluent which is pharmaceutically acceptable and iscompatible with the effective component(s). Preferable examples of thediluent include water, physiological saline, dextrose, glycerol,ethanol, and mixtures thereof. Further, if desired, the vaccinecomposition of the invention may include a little amount of an assistingsubstance such as a wetting agent or emulsifier and pH buffer.

The vaccine composition of the invention may be administered byinjection (including intradermic one, hypodermic one, and intramuscularone) via various routes (e.g. nasal cavity, mucosa, oral, intravaginal,urethra, and intraocular) or may be non-orally administered using apatch (endermic administration) or a suppository or the like.

The vaccine composition of the invention may be administered orally. Fororal administration, the vaccine composition of the invention is used incombination with a diluent such as pharmaceutical-grade mannitol,lactose, starch, magnesium stearate, sodium saccharate, cellulose, andmagnesium carbonate. The vaccine composition of the invention whenadministered orally may be in the form of a solution, a suspension, atablet, a pill, a capsule, a sustained release formulation, or a powdermedicine. In embodiments wherein the vaccine composition of theinvention is provided in a freeze-dried form, the freeze-dried substancemay be reconstructed as a suspension before administration. Thereconstruction is preferably carried out in a buffer solution.

The vaccine composition of the invention may contain an adjuvant forenhancing effectiveness of the vaccine. Examples of the effectiveadjuvant include, but not limited to, aluminum hydroxide, aluminumphosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica,kaoline, carbon, water-in-oil type emulsion, oil-in-water type emulsion,Muramyl dipeptide, bacterial endotoxin, fat X, Bordetella pertussis,Corynebacterium parvum, polyribonucleotide, sodium alginate, lanoline,lysolecithin, vitamin A, sponin, liposome, levamisole, DEAE-dextran,block copolymer, and other synthesized adjuvant. These adjuvants arecommercially available from various suppliers. Representative examplesof these adjuvants include an oil-in-water type adjuvant, an aluminumhydroxide adjuvant, and an adjuvant mixed with these adjuvants. Anadjuvant which is permitted to be applied to humans is aluminumhydroxide.

In another aspect, the invention provides a vaccine kit. The vaccine kitcorresponds to the aforementioned vaccine composition, and may includethe peptide of the invention or the polynucleotide of the invention asan effective vaccine component and may include anti-TNF, and may furtherinclude one or more optional components.

In certain embodiments, the vaccine kit of the invention includes thepeptide of the invention and/or peptide and anti-TNF. In anotherembodiment, the vaccine kit of the invention contains the polynucleotideof the invention and/or polynucleotide and anti-TNF. The polynucleotideof the invention is preferably in a form capable of expressing thepeptide encoded by the polynucleotide of the invention, and is morepreferably provided in a form of an expression vector to which thepolynucleotide of the invention is operably linked.

The kit typically includes a package with one or more containers (e.g.,bottle, plate, tube, and dish) having therein a specific material. Thekit preferably has directions for using the individual materialscontained in any one of individual containers constituting the kit.Further, the kit of the invention may be a package in which a pluralityof different compositions are contained. The form of the composition maybe as above, and if the composition is in the form of a solution, thecomposition may be contained in a container. The kit of the inventionmay be designed such that materials A and B are mixed and included in asingle container or are included in respective containers. The“directions” may be written on a paper or other kind of a medium or maybe printed out. Alternatively, the “directions” may be in the form of amagnetic tape or an electronic medium such as a computer-readable discor tape and a CD-ROM. Further, the kit of the invention may include acontainer containing a diluent, a solvent, a cleaning liquid, or otherreagent. Further, the kit may include beforehand an instrument necessaryfor prevention/treatment of acne caused by infection withPropionibacterium acnes.

Use of the vaccine of the invention as above enables efficientlypreventing/treating inflammation (acne) caused by infection withPropionibacterium acnes.

That is, a method for treating or preventing inflammation caused byPropionibacterium acnes, including the step of administering to anindividual a vaccine containing a therapeutically effective amount ofthe peptide of the invention or a therapeutically effective amount ofthe polynucleotide of the invention, and optionally a therapeuticallyeffective amount of anti-TNF.

The term “therapeutically effective amount” used herein indicates theamount of peptide or polynucleotide which amount enables the peptide orthe polynucleotide to exert a desired therapeutic effect or prophylacticeffect. In embodiments, wherein anti-TNF is present, the anti-TNF mayalso be present in a therapeutically effective amount. Thetherapeutically effective amount may differ depending on the age, theweight, and other health conditions of an individual to which thevaccine is administered, on the condition of an inflammation to betreated, and on a method of administration. In all instances, at itsmost basic level, the desired effect is a regression, reduction orelimination of tumor cells in tumor tissue of the patient when comparedto the tumor cells in the tumor tissue of the patient prior to employingthe therapy methods of the invention. In certain embodiments ofsubcutaneous administration, administration of a vaccine may be suchthat 5-10 μg of the peptide per kg weight or 10-50 μg of thepolynucleotide per kg weight is supplied in one administration. Toinduce an immune response sufficient for anti-inflammation, the vaccinemay be administered multiple times, two times or more, to the patient.In certain embodiments, administration is made at a predeterminedinterval.

Another example is as follows: in a case of a vaccine including amixture of MAP based on the peptide consisting of the amino acidsequence indicated by SEQ NO. 1 and MAP based on the peptide consistingof the amino acid sequence indicated by SEQ NO. 3 (weight ratio is 1:1),administration may be made via hypodermic injection in such a mannerthat 100-500 μg of each peptide per one shot is administered in the formof a physiological saline solution five times or so at an interval offrom three to five days.

In a case of administration via a patch, one dose may be small, thenumber of dose may be reduced, and an individual can treathimself/herself using the patch.

The term “patient or individual” used herein indicates individual humansand mammals other than humans (such as rats, mice, rabbits, pigs,cattle, and monkeys).

The invention provides an antibody which specifically recognizes any ofthe peptides of the invention. That is, an antibody of the invention canspecifically bind to any of the peptides. In certain embodiments, theantibody is preferably an antibody which binds to (1) the peptideconsisting of the amino acid sequence indicated by SEQ NO. 1 or 3 or (2)a multivalent antigen peptide synthesized by the MAP process from thepeptide consisting of the amino acid sequence indicated by SEQ NO. 1 or3.

The term “antibody” used herein indicates an immunoglobulin (IgA, IgD,IgE, IgG, IgM, and Fab fragments thereof, F(ab′)₂ fragment and Fcfragment). Examples of the antibody include, but not limited to,polyclonal antibodies, monoclonal antibodies, single-chain antibodies,and antiidiotype antibodies.

The antibody of the invention may be produced by conventional methodsknown in the field to which the invention pertains. For example, theantibody can be obtained by administering the peptide of the inventionto a living body of a mammal and causing an immune response so that anantibody is produced in the living body. The antibody obtained in thiscase is generally a polyclonal antibody. A monoclonal antibody can beproduced by conventional and known hybridoma techniques using thepeptide of the invention. Alternatively, the monoclonal antibody can beproduced by application of a recombinant DNA technique or chemicalsynthesis.

Use of the antibody of the invention enables one to easily purify thepeptide of the invention. For example, by subjecting the peptide of theinvention expressed in a cell using the vector of the invention or thetransformant of the invention to affinity purification using theantibody of the invention, it is possible to efficiently collect thepeptide.

The antibody of the invention may be any antibody provided the antibodyspecifically binds to the peptide of the invention. The antibody is notlimited in terms of the kinds of individual immunoglobulins (IgA, IgD,IgE, IgG, or IgM), a method for preparing a chimeric antibody, a methodfor preparing a peptide antigen and the like, which are specificallydescribed herein. Therefore, an antibody obtained by a method other thanthe above-described methods is also encompassed in the invention.

A method of the invention for determining effectiveness of a vaccineagainst Propionibacterium acnes is only required to include a detectionstep of detecting whether a polynucleotide encoding the above peptideexists or not in a sample collected from a living body, and is notparticularly limited in terms of other specific steps and instrumentsand devices to be used.

In a case where a polynucleotide encoding the peptide is detected in thedetection step, it can be determined that administration of the vaccineof the invention to a test subject is highly likely to result inefficient suppression of inflammation caused by Propionibacterium acnes.Specifically, when the polynucleotide encoding the above peptide isdetected, it is determined that the vaccine of the invention is likelyto be effective against Propionibacterium acnes.

The method for detecting the polynucleotide encoding the peptide is notparticularly limited. For example, the method may be detection by PCRamplification or detection by determining a base sequence of a PCRproduct. By determining the base sequence of the PCR product, it ispossible to determine more accurately whether a vaccination effect canbe exerted or not. Whether the vaccination effect can be exerted or notmay be determined also depending on whether a PCR fragment is generatedor not by amplification. For example, in a case where a fragment with adesired length is generated by amplification. using a specific primerfor amplifying the above polynucleotide (in a case where the result ofPCR detection is positive), it is highly possible that the test subjectis infected with Propionibacterium acnes retaining the abovepolynucleotide, and so it is determined that the vaccination effect ishighly expected. On the other hand, in a case where a fragment with adesired length is not generated (in a case where the result of PCRdetection is negative), it is determined that the vaccination effect isexpected little. Before PCR amplification, Propionibacterium acnes thatcan be contained in a sample collected from the test subject may begrown by culturing the sample.

A method for determining whether the vaccine of the invention againstPropionibacterium acnes is to be used or not may be made by detectingwhether a partial sequence of 16S rDNA of Propionibacterium acnes in asample collected from a living body exists or not. Detection of whetherthe partial sequence of 16S rDNA exists or not may be detection by PCRamplification and detection by determining a base sequence of a PCRproduct. This determination may indicate that the inflammation of thetest subject is derived from Propionibacterium acnes. This may be abasis for using the vaccine.

A model animal which is a mammal other than humans, such as mice inlater-mentioned Examples, whose abdominal midline was intradermicallyinjected with Propionibacterium acnes with predetermined bacterial cellconcentration or more to cause inflammation of Propionibacterium acnes,and a method for preparing the model animal, are also encompassed in theinvention. In the model animal, inflammation of Propionibacterium acnesis created in a more efficient manner than a conventional method forcreating inflammation. A determination system having been used so far indeveloping an anti-acne drug is a one based on the assumption that thedrug is applied. Consequently, in developing a vaccine, there has beenconstructed so far no assured determination system which can determinethe effect of the vaccine in vivo. The method for determiningeffectiveness of the vaccine, the model animal, and the method forpreparing the model animal can be preferably used in developing avaccine against Propionibacterium acnes.

The following explains embodiments of the invention in more detail, withreference to Examples. It should be noted that the invention is notlimited to the Examples below and may be modified in terms of itsdetails. The invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. An embodiment based on a proper combination oftechnical means disclosed in different embodiments is encompassed in thetechnical scope of the invention. All the documents cited in thespecification are incorporated by reference.

EXAMPLES Example 1—Culture of Propionibacterium Acnes

Strains of Propionibacterium acnes (hereinafter abbreviated as “P.acnes”) (ATCC6919) were purchased from RIKEN BioResource Center (JCMCatalogue No. 6425). The strains of the purchased P. acnes were culturedaccording to culture condition information from the center.Specifically, in an anaerobic glove box, the strains of the P. acneswere suspended in 100 ml of a GAM culture medium (manufactured by NipponSuisan Kaisha, Ltd.) and cultured anaerobically using a vial at 37° C.for three days.

Example 2—Causing Intradermical Inflammation in Mouse Skin by P. Acnes

Living bacterial cells of P. acnes were put in a deaerated physiologicalsaline while keeping an anaerobic condition to prepare a suspension withthe number of the living bacterial cells of P. acnes of 5×10⁷ cells/ml,1×10⁸ cells/ml, or 5×10⁸ cells/ml. The anaerobic condition was kept byspraying a nitrogen gas. The number of the living bacterial cells of P.acnes was calculated based on the assumption that OD measurement valueof 0.98-1.02 at 550 nm measured by a spectrophotometer corresponds to5×10⁸ cells/ml, with reference to Ramstad S. et al., Photochem.Photobiol. Sci., 2006, 5, 66-72.

50 μl of the prepared suspension of P. acnes was intradermicallyinjected into shaven abdominal midlines, neck portions, lumbar dorsalportions, tail root dorsal portions, and auricles of male Balb/c mice(9-10 weeks old). The inflammatory areas (major axis×minor axis: mm²) atindividual portions to which the suspension was injected intradermicallywere measured at predetermined times. Each group consisted of fiveindividuals.

As a result, in a group to which the suspension of P. acnes whosebacterial cell concentration was 5×10⁸ cells/ml was injected,inflammation was apparently confirmed. FIG. 1 is a graph showing theresult of inflammatory areas at individual portions observed five daysafter the injection. As illustrated in FIG. 1, inflammation was observedin individual portions to which the suspension was intradermicallyinjected, and the inflammatory area was largest at an intradermicportion of the abdominal midline. That is, when causing intradermicinflammation of mice, intradermic injection into the abdominal midlinewas most effective. Inflammation at the auricle was the mildest amongthe observed portions. FIG. 2 is a graph showing the result of theinflammatory area at the abdominal midline at individual times after thesuspension was intradermically injected into of the abdominal midline.As illustrated in FIG. 2, the inflammatory area at the abdominal midlinewas largest five days after the injection of P. acnes.

In contrast thereto, in the groups to which the suspensions of P. acneswhose bacterial cell concentrations were 5×10⁷ cells/ml and 1×10⁸cells/ml, respectively, were injected, no inflammation could be causedat any portions and at any times.

Example 3—Preparation of Peptide

The powder of PepA which was a peptide consisting of the amino acidsequence indicated by SEQ NO. 1 or the powder of PepD which was apeptide consisting of the amino acid sequence indicated by SEQ NO. 3 wasdissolved in physiological saline (produced by Otsuka PharmaceuticalCo., Ltd.) to prepare 800 μg/ml of a peptide solution.

As Comparative Examples, there were used PepB which was a peptidecorresponding to 10th-26th amino acid residues in a membrane protein ofPropionibacterium acnes which consists of the amino acid sequenceregistered in Genbank/EMBL/DDBJ accession No. AAT81852, and PepC whichwas a peptide corresponding to 145th-166th amino acid residues in amembrane protein of Propionibacterium acnes which consists of the aminoacid sequence registered in Genbank/EMBL/DDBJ accession No. YP_055518.Table 1 shows information on the amino acid sequence in individualpeptides. The sequences used as the Comparative Examples were amino acidsequences which may be strong candidates for antigens in producingantibodies, i.e. amino acid sequences which are abundant in hydrophilicamino acids and charges, and were selected from amino acid sequenceswith a turn structure. Further, the proteins consisting of the aminoacid sequences registered in the above accession Nos. are proteinssupposed to be expressed as a result of genome analysis described inBruggemann H. et al., “The Complete Genome Sequence of PropionibacteriumAcnes, a Commensal of Human Skin,” Science, 2004, 305, 671-673.

TABLE I Accession No. Peptide SEQ Derived Name Amino Acid Sequence No.Protein PepA  ⁹⁰AIQEKYGDDRERAG¹⁰³  1 AAT84059 PepB ¹⁰GRKPDTNKRSWHRKASR²⁶ 11 AAT81852 PepC ¹⁴⁵IDQVREYRHRDDDDDEDPGEDG¹⁶⁶ 12YP_055518 PepD ²⁶⁰KDADKDNPTYQKV²⁷²  3 YP_056445

Example 4—Anti-Inflammatory Effect of Peptide PepA Against InflammationCaused by P. Acnes

In Example 2, the area of the inflammation caused by P. acnes waslargest when five days had passed from intradermic injection of 50 μl ofP. acnes (5×10⁸ cells/ml) into the abdominal midline. Accordingly, theanti-inflammatory effects of individual peptides were determined underthe same conditions, i.e. by intradermically injecting 50 μl of P. acnes(5×10⁸ cells/ml) into the abdominal midline and measuring theinflammatory area (mm²) five days after the injection. FIG. 3illustrates the procedure for examining the anti-inflammatory effect.

FIG. 3 is a drawing schematically illustrating the procedure forexamining the anti-inflammatory effect. As illustrated in FIG. 3, PepA(40 μg/50 μl/one individual) was intradermically vaccinated into dorsalportions of male Balb/c mice (7 weeks old at the first vaccination ofPepA) 18 days before the injection of living bacterial cells of P. acnes(Day “0” in FIG. 3), 11 days before the injection (Day “7” in FIG. 3),and 4 days before the injection (Day “14” in FIG. 3), i.e. three timesin total. Then, P. acnes was intradermically injected into the abdominalmidlines of the mice, and the inflammatory areas were measured 5 daysafter the injection (Day “23” in FIG. 3). Each group consisted of 10mice. As Comparative Examples, there were prepared a group of mice towhich PBS without PepA was administered and a group of mice to whichPepB or PepC was administered instead of PepA, and P. acnes was injectedinto the groups similarly. These results are shown in FIG. 4.

FIG. 4 is a graph showing the result of the anti-inflammatory effect ofPepA. As illustrated in FIG. 4, the group to which PepA was administeredexhibited a statistically significantly reduced inflammatory area(P=0.00865 (P<0.01)), compared with the control group to which PBS wasadministered. That is, PepA statistically significantly exhibits theanti-inflammatory effect against the inflammation caused by P. acnes. Inthe case of the group to which PepB was administered, P=1. In the caseof the group to which PepC was administered, P=0.1655. These numeralswere not significant statistically, and so it was considered that PepBand PepC do not exhibit the anti-inflammatory effect against theinflammation caused by P. acnes.

The above results show that the composition containing the peptide PepAis effective as a vaccine against P. acnes, i.e. an acne vaccine.

Example 5—Anti-Inflammatory Effect of Peptides PepA and PepD AgainstInflammation Caused by P. Acnes

The anti-inflammatory effects of individual peptides were examined usingan inflammation causing system under the same conditions as in Example 4except that the peptides to be administered to mice before injection ofP. acnes were changed from PepA, PepB, and PepC to PepA and PepD. Theresults are shown in FIG. 5.

FIG. 5 is a graph showing the results of the anti-inflammatory effectsof PepA and PepD. As shown in FIG. 5, the group to which PepA wasadministered exhibited a statistically significantly reducedinflammatory area (P=0.0012 (P<0.01)), compared with the control groupto which PBS was administered, as in the result of Example 4. Further,the group to which PepD was administered exhibited a statisticallysignificantly reduced inflammatory area (P=0.00175 (P<0.01)), comparedwith the control group to which PBS was administered.

The above results show that the composition containing the peptide PepAor PepD is effective as a vaccine against P. acnes, i.e. an acnevaccine.

Example 6—Identification of Strain Harbored by Acne Patient Extractionof 16S rDNA

Purulence collected from a diseased part of an acne patient was culturedanaerobically on a GAM flat plate agar medium at 37° C., and a part ofthe bacterial cells was collected. The collected bacterial cells weretransferred to a GAM flat plate agar medium and cultured anaerobicallyso that a single colony was produced. Bacterial cells from the singlecolony were collected, and suspended in a 20 mM NaOH aqueous solution.The suspension was heated at 94° C. for 3 min, the bacterial cells werelysed, and 16S rDNA was extracted. Using the extracted 16S rDNA, a basesequence of 16S rDNA of the bacterial cells with which the patient wasinfected was determined. The base sequence was determined with respectto each of three sets of bacterial cells collected from three acnepatients, respectively.

Determination of Base Sequence of 16S rDNA

The extracted 16S rDNA was used as a template DNA. Further, as a primer,there were used primer 9F (forward primer): 5′-GTTTGATCCTGGCTCA-3′ (SEQNO. 5) and primer 800R (reverse primer): 5′-TACCAGGGTATCTAATCC-3′ (SEQNO. 6). PCR carried out here consisted of 30 cycles each consisting of astep at 94° C. for 30 seconds, a step at 55° C. for 60 seconds, and astep at 72° C. for 60 seconds. The PCR was carried out by AmpliTaq GoldDNA Polymerase (manufactured by Applied Biosystems). After the reaction,a PCR product was purified. Subsequently, a cycle sequence reaction wascarried out using the purified PCR product. A product obtained in thecycle sequence reaction was purified, supplied to an ABI PRISM 310Genetic Analyzer System (manufactured by Applied Biosystems), and a basesequence was determined using analysis software BioEdit. The primersused in the cycle sequence reaction were the above primer 9F and primer536R: 5′-GTATTACCGCGGCTGCTGG-3′ (SEQ NO. 7). The cycle sequence reactionconsisted of 25 cycles each consisting of a step at 96° C. for 10seconds, a step at 50° C. for 5 seconds, and a step at 60° C. for 4minutes.

Result of Determining Base Sequence

The result of determining the base sequence showed that in each of threeseparated bacterial strains that were established, derived from patients(patient strains 1-3), the base sequence of 16S rDNA at an amplifiedpart (SEQ NO. 16) had the same sequence as that of the correspondingbase sequence in ATCC6919 strain of P. acnes (SEQ NO. 8). FIG. 6 showsthe result with respect to the patient strain 1. Accordingly, the threeseparated bacterial cells derived from the patients were identified asP. acnes.

Example 7—Anti-Inflammatory Effect of Peptide PepA Against InflammationCaused by P. Acnes Derived from Patient

The anti-inflammatory effect of PepA was examined by the same procedureas in Example 5 except that bacterial cells of P. acnes to be injectedinto mice were the acne-patient-derived bacterial cells of P. acneswhich were isolated in Example 6. The result is shown in FIG. 7.

FIG. 7 is a graph showing the result of intradermically injecting, afterimmunization by PepA, the patient strains 1-3 (P1-P. acnes to P3-P.acnes) to the abdominal midlines of mice and measuring inflammatoryareas of the mice 5 days after the injection. As shown in FIG. 7, thegroup to which PepA was administered showed P=0.0252 (P<0.05) withrespect to the patient strain 1, P=0.0476 (P<0.05) with respect to thepatient strain 2, and P=0.0237 (P<0.05) with respect to the patientstrain 3, compared with the control group to which PBS was administeredinstead of PepA. That is, administration of PepA enabled statisticallysignificantly reducing an inflammatory area with respect to injection ofeach of the patient strains. This suggests that PepA is widely effectiveas an acne vaccine.

Example 8—Examination of Base Sequence Encoding PepA or PepD in P. AcnesDerived from Acne Patient

In order that PepA or PepD serves effectively as a vaccine, it isdesirable that target P. acnes strains have a protein containing PepA orPepD. Accordingly, whether the target P. acnes strains had a proteincontaining PepA or PepD was examined by carrying out PCR and bydetermining and checking a base sequence of PCR products.

Confirmation of PepA by PCR

Primers used here for detecting a base sequence encoding an amino acidsequence of PepA were primer L: 5′-GATGAAAGCCATCCAGGAAA-3′ (SEQ NO. 9)and primer R: 5′-GCACACGAAACAACGCTAGA-3′ (SEQ NO. 10). PCR carried outhere consisted of 35 cycles each consisting of a step at 95° C. for 15seconds, a step at 60° C. for 20 seconds, and a step at 72° C. for 30seconds. The PCR was carried out using AmpliTaq Gold DNA Polymerase(manufactured by Applied Biosystems).

As a result, a PCR fragment with a desired length was obtained byamplification from the detected patient-derived P. acnes, andconsequently existence of a base sequence encoding the amino acidsequence of PepA was deduced. This suggests that a protein correspondingto PepA has the same amino acid sequence as that of PepA.

Further, specimens collected from 21 volunteers were examined as towhether they had a base sequence encoding the amino acid sequence ofPepA, and 19 cases out of 21 cases were positive. The term “positive”used in the present Examples indicates that a fragment with a desiredlength was generated by PCR amplification using a specific primer. Theterm “negative” used in the present Examples indicates that a fragmentwith a desired length could not be generated by PCR amplification usinga specific primer.

Similarly, the specimens collected from the 21 volunteers were examinedby PCR as to whether they had 16S rDNA of P. acnes. The result showedthat the 19 cases being positive in examination of the base sequenceencoding the amino acid sequence of PepA was also positive inexamination of 16S rDNA, and the 2 cases being negative in examinationof the base sequence encoding the amino acid sequence of PepA was alsonegative in examination of 16S rDNA. The results of the 2 cases beingnegative in PCR detection for confirming the base sequence encoding theamino acid sequence of PepA do not indicate that the base sequenceencoding the amino acid sequence of PepA was not carried by the 2volunteers but indicates that the number of bacterial cells of P. acneswas so low that the bacterial cells could not be detected. From theabove results, it was suggested that a protein containing the amino acidsequence of PepA exists in the 19 cases in which 16S rDNA of P. acneswas confirmed.

TABLE 2 Mean PCR Subjects Total Age Mongoloid Caucasoid Positives (%)Male 4 25.75 2 2  4 (100) Female 15 29.43 14 1 15 (100) Total 19 28.6116 3 19 (100) PCR 19 (100) 16 (100) 3 (100) Positives (%)

Further, similarly as above, specimens collected from another 11volunteers were examined by PCR as to whether they had 16S rDNA of P.acnes. The result showed that each of the specimens was P. acnespositive. Further, the 11 cases were examined as to whether they had abase sequence encoding the amino acid sequence of PepA and all of the 11cases were found to be positive. Table 3 shows the results of the 11cases together with the results of the 19 cases which were found to bepositive.

TABLE 3 Mean PCR Subjects Total Age Mongoloid Caucasoid Positives (%)Male 7 25.86 5 2  7 (100) Female 23 29.55 22 1 23 (100) Total 30 28.6627 3 30 (100) PCR 30 (100) 27 (100) 3 (100) 30 (100) Positives (%)

Confirmation of Base Sequences Encoding PepA and PepD

With respect to each of specimens collected from 5 volunteers and P.acnes described in Example 1, DNA encoding the amino acid sequence ofPepA and DNA encoding the amino acid sequence of PepD were amplified byPCR. In amplifying DNA encoding PepA, the primer L (SEQ NO. 9) and theprimer R (SEQ NO. 10) were used as primers. In amplifying DNA encodingPepD, primer om1L: 5′-GGTGCTGTCGTCAATAACAACTTC-3′ (SEQ NO. 14) andprimer om1R: 5′-GGAGTGGCCAGAGACGATCT-3′ (SEQ NO. 15) were used asprimers. PCR carried out here consisted of (i) a step at 95° C. for 5minutes, thereafter (ii) 35 cycles each consisting of a step at 95° C.for 15 seconds, a step at 53° C. for 20 seconds, and a step at 72° C.for 30 seconds, and thereafter (iii) a step at 72° C. for 7 minutes anda step at 25° C. for 10 minutes. Table 4 shows the result of PCR.

As shown in Table 4, in all the specimens, with respect to PepA, PCRproducts with a desired size were generated by amplification. The caseswhere the PCR products with a desired size were generated byamplification are shown as “positive”. With respect to PepD, PCRproducts with a desired size were generated by amplification except forthe specimen of the patient strain D.

Subsequently, base sequences of the PCR products were determined todetermine deduced amino acid sequences. Determination of the basesequences was made by using 3130x1 Genetic Analyzer, Sequencing AnalysisSoftware ver 5.3.1., and KB BaseCaller (each manufactured by AppliedBiosystems). Table 4 shows the result. As shown in Table 4, in all thespecimens, the sequence of PepA was conserved. That is, the deducedamino acid sequence thus obtained was the same as the amino acidsequence indicated by SEQ NO. 1. On the other hand, in the specimens ofthe patient strains C and E, the sequence of PepD was conserved. Thatis, the deduced amino acid sequence thus obtained was the same as theamino acid sequence indicated by SEQ NO. 3. In the specimens of thepatient strains A and B, the deduced amino acid sequence had onemutation of amino acid. Specifically, an amino acid residue positioned11.sup.th from N-terminus of the amino acid sequence indicated by SEQNO. 3 was changed from Gln to Lys. This mutation was also observed instrains derived from Riken BioResource Center which were used todetermine the anti-inflammatory effect of individual peptides in theExamples. The patient strain A is a specimen derived from the acnepatient in the Example 6.

TABLE 4 PCR Amino Acid Sequence Specimen PepA PepD PepA PepD Stainderived Positive Positive Sequence Partial Mutation From RikenConservation (¹¹Gln→Lys) (JCM6425) Patient strain Positive PositiveSequence Partial Mutation A Conservation (¹¹Gln→Lys) Patient strainPositive Positive Sequence Partial Mutation B Conservation (¹¹Gln→Lys)Patient strain Positive Positive Sequence Sequence C ConservationConservation Patient strain Positive Negative Sequence D ConservationPatient strain Positive Positive Sequence Sequence E ConservationConservation

However, as shown in the Example 5 and FIG. 5, single administration ofPepD can yield an anti-inflammatory effect on inflammation caused bystrains derived from RIKEN BioResource Center which contain mutation inthe amino acid sequence of PepD. This shows that PepD can serve as avaccine effectively even on inflammation caused by strains containingPepD having mutation of one amino acid.

Similarly, in P. acnes in the specimens collected from the 30 volunteersshown in Table 5, DNA encoding the amino acid sequence of PepA and DNAencoding the amino acid sequence of PepD were amplified by PCR and basesequences of respective PCR products were determined, so that respectivededuced amino acid sequences were determined. Table 5 shows the results.

TABLE 5 Number of Number of Cases of Cases of Number of Complete PartialCases of PCR Detection Number of Sequence Sequence Unidentified (Simple)Cases Conservation Mutation Sequende PepA 30 30 0 0 Positive PepA 0 0 00 Negative PepD 26 20 6 (¹¹Gln→Lys 0 Positive in all cases) PepD 4 0 0 4Negative

As shown in Table 5, in all the specimens examined here, the amino acidsequence of PepA was conserved completely. The results of Tables 4 and 5show that the PepA sequence of actual patient-derived P. acnes wasconserved very well.

On the other hand, in the 20 cases out of the 30 cases examined, theamino acid sequence of PepD was conserved completely (66.7%). In the 6cases out of the cases examined, the amino acid sequence was partiallymutated (¹¹Gln→Lys). This is a partial mutation equal to that of theRIKEN BioResource Center strains to which administration of a vaccinewas found to be effective by the test using mice. Accordingly, in viewof the effectiveness of a vaccine effect, the PepD sequence is supposedto be effective in 26 cases out of the 30 cases (86.7%). That is, thePepD sequence was conserved well.

In view of the above, it is considered that PepA and PepD have sequenceswhich are well conserved in bacterial cells of patients, and thereforeare highly effective as vaccines for patients.

Example 9—Vaccine Use Against P. Acnes Preparation of Vaccine

As described above, it was found in the acne inflammatory causing testsystem using mice in the Example 5 that two kinds of single chainpeptides (PepA and PepD) have an anti-inflammatory effect. In order thatthese peptides work more effectively, MAP peptides for PepA and PepDwere synthesized by the MAP process. The synthesis peptide thus obtainedconsisted of 7-13 molecules of the single chain peptide of PepA or PepDbound to a MAP structure. A MAP peptide mixture solution in which theMAP peptides for PepA and PepD, respectively, were mixed was preparedand used as a vaccine.

Preparation of the mixture solution was made as follows. Initially,individual MAP peptides were prepared, and then dialyzed with distilledwater, and freeze-dried and powdered. Subsequently, respective powderswere dissolved in physiological saline (produced by OtsukaPharmaceutical Co., Ltd.) so that concentrations of the respective MAPpeptides were 1 mg/ml (each of the resulting solutions are hereinafterreferred to as “PA-MAP solution”). The PA-MAP solutions were subjectedto an endotoxin examination and a cytotoxic examination. The amount ofendotoxin met the allowable amount (5 EU or less/kg weight/1 shot)according to NIH. Further, in the cytotoxic examination, 10 μl of a testpeptide solution and 90 μl of peripheral-blood mononuclear cells (2×10⁶cells/ml in plain RPMI 1640) were co-cultured at 37° C. for 24 hours.The cell survival ratio was substantially equal (with difference of+/−5% or less) to that of a control in which peripheral-bloodmononuclear cells to which physiological saline was added were cultured,and consequently no toxicity was found.

Case 1

100 μl of the prepared PA-MAP solution was hypodermically vaccinatedinto an upper arm of the acne patient five times at an interval of fivedays. The acne patient was a patient carrying the patient strain A inthe Example 8. FIG. 8 shows the result of observation.

As shown in FIG. 8, before the treatment (at first administration),whole areas of skin bulged and elevated (indicated by arrows in thedrawing) due to hypodermic proliferation of acne bacterium, accompaniedby inflammation. One month after the first vaccination, although bulgeand elevation of acne were still observed, they were divided into twoareas and inflammation was improved and localized. Three months afterthe last vaccination, inflammation was decreasing gradually, acne wasturning into scars, and the bulge and elevation were becoming flat.Since it was in the course of the treatment, pigmentary deposit wasobserved. There was observed no malign phenomenon caused by vaccination.

Case 2

A specimen was collected from the acne patient carrying the patientstrain C in the Example 8, and it was confirmed whether the acne patientwas infected with P. acnes or not by amplifying 16S rDNA of P. acnes byPCR. The result showed that the acne patient was P. acnes positive.Amplification of 16S rDNA of P. acnes was carried out using (i) primerL1 consisting of the same sequence as the primer 9F and (ii) primer R2:5′-GCACGTAGTTAGCCGGTGCT-3′ (SEQ NO. 13) by PCR consisted, of (i) a stepat 95° C. for 5 minutes, and thereafter (ii) 35 cycles each consistingof a step at 95° C. for 15 seconds, a step at 55° C. for 20 seconds, anda step at 72° C. for 30 seconds and (iii) a step at 72° C. for sevenminutes.

300 μl of the prepared PA-MAP solution was hypodermically vaccinatedinto an upper arm of the acne patient five times at an interval of threedays. FIG. 9 shows the result of observation.

As shown in FIG. 9, whole areas of inflammation on the cheek observedbefore the treatment (at first administration) were improved one monthand three months after the first vaccination. There was observed nomalign phenomenon caused by vaccination.

Case 3

A specimen was collected from the acne patient carrying the patientstrain D in the Example 8, and it was confirmed whether the acne patientwas infected with P. acnes or not by amplifying 16S rDNA of P. acnes byPCR under the same conditions as those in the Case 2. The result showedthat the acne patient was P. acnes positive.

300 μl of the prepared PA-MAP solution was hypodermically vaccinatedinto an upper arm of the acne patient five times at an interval of threedays. FIG. 10 shows the result of observation.

As shown in FIG. 10, whole areas of inflammation around the noseobserved before the treatment (at first administration) were improvedone month and three months after the first vaccination. There wasobserved no malign phenomenon caused by vaccination.

Case 4

A specimen was collected from the acne patient carrying the patientstrain E in the Example 8, and it was confirmed whether the acne patientwas infected with P. acnes or not by amplifying 16S rDNA of P. acnes byPCR under the same conditions as those in the Case 2. The result showedthat the acne patient was P. acnes positive.

300 μl of the prepared PA-MAP solution was hypodermically vaccinatedinto an upper arm of the acne patient five times at an interval of fivedays. FIG. 11 shows the result of observation.

As shown in FIG. 11, whole areas of inflammation on wide areas of thecheek observed before the treatment (at first administration) wereimproved one month and three months after the first vaccination. Therewas observed no malign phenomenon caused by vaccination.

The above results show that the composition containing the peptide PepAand the peptide PepD is effective as a vaccine against P. acnes, i.e. anacne vaccine.

Use of the invention enables an immunotherapy for prevention andtreatment of acne. Accordingly, the invention is applicable to medicalfields, pharmaceutical fields etc. and is very useful.

Example 10—Preparation of Acne Vaccine Including Anti-TNF

A vaccine in accordance with the invention was prepared by combining 500μg of peptide antigen ABF155 and 500 μg of peptide antigen ABF156 with50 mg of anti-TNF. The anti-TNF was commercially obtained under thetrade name Enbrel. A control vaccine was prepared by combining 500 μg ofpeptide antigen ABF155 and 500 μg of peptide antigen ABF156 withoutanti-TNF. Each of the vaccines was administered by subcutaneousinjection to patients once per week for a total of twelve weeks. Thefollowing blood tests were administered once per month for a total offour months: WBC, RBC, Hb, Hemogram, biochemical tests, IgM, IgG, IgGsubtype. Propioni Bacterium Antibody Titer (ELISA assay) and CytokineAssays were obtained. The assays included the measurement of antibodytiters and T-cell responses to the p-acnes bacteria and vaccine peptidesemploying peripheral blood samples obtained from the patients before andafter receipt of the vaccine.

Example 11—Preparation and Administration of Acne Vaccine IncludingAnti-TNF

A total of eight vaccines were prepared in accordance with the inventionand each of the vaccines was administered to a different patient whichwas inflicted with Propionibacterium acnes. Administration consisted ofinjecting the vaccine into the hip of each patient once per weekstarting at week 0. Thus, every week subsequent to the initialinjection, another injection was administered in the hip of the patient.Photographs were taken at week 0 of an infected area on each of theeight patients and subsequent photographs were taken every four weeksthereafter. The photographs were used to identify the initial number ofacne spots and the progression and/or regression of the acne spots overtime in response to the vaccine. In addition, blood tests were conductedat week 0 for each of the eight patients and subsequent blood tests wereconducted every four weeks thereafter.

Six of the vaccines included the combination of peptide and anti-TNF,and two of the vaccines included peptide only, i.e., in the absence ofanti-TNF. At week 0, the initial vaccines, the six peptide/anti-TNFvaccines were prepared by combining 500 μg of peptide antigen ABF155 and500 μg of peptide antigen ABF156 with 50 mg of anti-TNF (commerciallyobtained under the trade name Enbrel). At week 0, the initial vaccines,two control vaccines were prepared by combining 500 μg of peptideantigen ABF155 and 500 μg of peptide antigen ABF156 without anti-TNF.For each of the vaccines administered after week 0, the amount ofpeptide and anti-TNF remained the same. However, it was contemplatedthat the amount of anti-TNF in each of the vaccines administered afterweek 0 may be adjusted in accordance with the patients' overallconditions. For example, the original vaccine consisted of 50 mg ofanti-TNF and it was contemplated that subsequent injections may havecontained either the same, more or less than this amount. In certainembodiments, wherein improvement is observed, e.g., a regression of acnespots, a lower amount of anti-TNF may have been used and wherein thepatient's condition is observed to worsen, e.g., an increase in thenumber of acne spots, a higher amount of anti-TNF may have been used.

The data for each of patients #1 through #8 is shown in Tables A throughH, respectively. As shown in Tables A and B (for patients #1 and #2),the administration of the vaccine including peptide and anti-TNF to theacne patients resulted in a complete response. The number of acne spotswere significantly reduced (from 8 spots at week 0 to 0 spots at week12). As shown in Table C (for patient #3), the administration of thevaccine including peptide only to the acne patient resulted in a stabledisease response for weeks 0 to 4. The number of acne spots basicallyremained the same (from 4 spots at week 1 to 4 spots at week 4).However, the injections for the next 12 weeks, i.e., weeks 4 through 16,employed a vaccine with anti-TNF. As a result, the number of acne spotswere reduced overall this 12 week period (from 4 spots at week 4 to 0spots at week 16). As shown in Table D (for patient #4), theadministration of the vaccine including peptide and anti-TNF to the acnepatient resulted in a partial response. The number of acne spots weresignificantly reduced (from 7 spots at week 0 to 3 spots at week 10). Asshown in Table E (for patient #5), the administration of the vaccineincluding peptide only to the acne patient resulted in a partialresponse. The number of acne spots were reduced (from 5 spots at week 0to 1 spot at week 11). As shown in Tables F and G (for patients #6 and#7), the administration of the vaccine including peptide and anti-TNF tothe acne patients resulted in a partial response. The number of acnespots for patients #6 and #7 were reduced (from 14 spots at week 0 to 2spots at week 12 for patient #6 and from 2 spots at week 0 to 1 spot atweek 12 for patient #7). The number of acne spots for As shown in TableH (for patient #8), the administration of the vaccine including peptideand anti-TNF to the acne patient resulted in a stable disease response(the number of spots at week 0 and week 12 was the same, i.e., 5 spots).

Based on the results, it is contemplated that the peptides present inthe vaccines were effective as antigen and the anti-TNF was effective tosuppress inflammation over-induced by the immune response which wastriggered by administration of the vaccine. Further, it was illustratedthat the combination of the peptides and anti-TNF was effective enhancevaccine efficacy.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

1. A composition to reduce inflammation in a patient with aPropionibacterium acnes infection, comprising: a peptide consisting ofan amino acid sequence of SEQ NO. 3; and a TNF inhibitor.
 2. Thecomposition of claim 1, wherein the peptide is in a form of amultivalent antigen peptide obtained by binding a plurality of thepeptide consisting of the amino acid sequence of SEQ NO. 3 by a linker.3. A method of suppressing inflammation caused by infection withPropionibacterium acnes in a patient, comprising: introducing into thepatient a peptide consisting of an amino acid sequence of SEQ NO. 3; andintroducing into the patient a TNF inhibitor.
 4. The method of claim 3,wherein the peptide and the TNF inhibitor are introduced simultaneously.5. The method of claim 4, wherein the peptide and the TNF inhibitor arein the form of a composition.
 6. The method of claim 3, wherein thepeptide and the TNF inhibitor are introduced sequentially.
 7. A vaccineto reduce inflammation in a patient with a Propionibacterium acnesinfection, comprising: a peptide consisting of an amino acid sequence ofSEQ NO. 3; and a TNF inhibitor.
 8. The vaccine of claim 7, wherein thepeptide is in a form of a multivalent antigen peptide obtained bybinding a plurality of the peptide consisting of the amino acid sequenceof SEQ NO. 3 by a linker.
 9. The vaccine of claim 8, wherein the peptideand the TNF inhibitor are combined in the form of a composition.
 10. Amethod of suppressing inflammation caused by infection withPropionibacterium acnes in a patient, comprising: introducing into thepatient a peptide consisting of an amino acid sequence of SEQ NO. 1; andintroducing into the patient a TNF inhibitor.
 11. The method of claim10, wherein the peptide and the TNF inhibitor are introducedsimultaneously.
 12. The method of claim 11, wherein the peptide and theTNF inhibitor are in the form of a composition.
 13. The method of claim10, wherein the peptide and the TNF inhibitor are introducedsequentially.